The overall objective of this application is to study the mechanism of hepatitis C virus (HCV) RNA replication and to establish a cell culture system for HCV RNA replication. Currently, the lack of an efficient replication system in tissue culture is one of the major obstacles in studying HCV. Our laboratory has expressed a recombinant HCV RNA polymerase, which is capable of replicating HCV RNA faithfully and appears independent of primers. Thus, this polymerase has the attributes expected of an authentic viral polymerase. This polymerase provides the opportunity to study HCV RNA replication in vitro and in vivo. Furthermore, it may allow the establishment of an HCV replication system in tissue culture. The following projects will be pursued: 1) Characterization of the HCV RNA polymerase activity in vitro. We will study the cis-acting sequences required for both (-)- and (+)-strand RNA synthesis in vitro, and the effects of the variable sequence at the 3'-UTR on RNA synthesis. We will also characterize the RNA products and the possible cellular factors on RNA synthesis in vitro. 2) Expression of a functional RNA polymerase in mammalian cells and establishment of an RNA replication system in cell culture. We will characterize the properties of the HCV polymerase expressed in mammalian cells. Once this polymerase is found to be active, we will use it as the basis for establishing an RNA replication system in cell culture, using an HCV defective-interfering (DI) RNA construct and full-length HCV RNA. 3) Characterization of the components of HCV RNA polymerase complex in the cells. We will study the interactions of other viral proteins, including ns4b and ns5a, with HCV polymerase. We will also characterize cellular proteins interacting with the polymerase. Finally we will study the effects of ns4b and ns5a on RNA synthesis in vivo.